DNA vector and transformed tumor cell vaccines

ABSTRACT

Customized whole cell cancer vaccines can be produced from autologous (ex vivo or in situ) or allogeneic human or veterinary patient cell lines. Cells are transformed with S. pyogenes DNA that expresses an Emm protein on the cell surface and cytosol. Treatment of cancer patients with an Emm vector vaccine induces an immunologic response to the cancer by enhancing immunogenicity of a tumor. Emm vaccines can be used in patients where the cancer is not identified due to lower tumor burden or used to treat a specific cancer and subsequently treat for a second type that may have arisen through metastasis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.15/418,798, filed Jan. 30, 2017, now U.S. Pat. No. 9,839,680, which is adivisional of U.S. application Ser. No. 15/110,248, filed Jul. 7, 2016,now U.S. Pat. No. 9,555,088, which is the U.S. national stageapplication of International Patent Application No. PCT/US2015/018688,filed Mar. 4, 2015, which claims the benefit of U.S. Provisional PatentApplication No. 61/948,980, filed Mar. 6, 2014.

The Sequence Listing for this application is labeled“Seq-List-replace.txt” which was created on Jun. 18, 2019 and is 21 KB.The entire content of the sequence listing is incorporated herein byreference in its entirety.

The field of the invention relates to therapeutic DNA vector vaccinesand particularly to cancer cells transformed in vitro or in vivo withthe vector. When the vector is administered in vivo into solid cancers,the polypeptide expressed by the vector enhances or augments an immuneresponse to targeted cancers. The DNA vectors function as vaccines whenadministered directly to solid cancers or after transformation ofisolated cancer cells prepared in vitro from a cancer patient areinjected into the patient as whole cell vaccines to treat a broad rangeof cancers, including multiple liquid and solid tumors.

BACKGROUND OF THE INVENTION

Multiple treatment modalities are available for treating cancer. Theyinclude surgical resection of the tumor, chemotherapy, radio therapiesand combination therapies. While resection can be curative, tumors recurin most cases. Chemotherapies utilize drugs that kill tumor cells byintercalation of DNA, inhibition of replication, or prevention ofmicrotubule assembly. To avoid killing healthy cells, a balance must beachieved by fine tuning the chemotherapy doses and regimens, whichshould be based on the type of tumor, stage, grade and overall tumorburden. Radiotherapy is essentially geared to kill cancer cells bydamaging DNA. Both chemo and radiotherapies result in a number of sideeffects in patients, including resistance to therapies, and in mostcases, tumors recur.

Conventional treatments for solid and circulating and liquid cancerstypically include chemotherapy and/or surgery. Recently there has beeninterest in developing vaccines in an effort to stimulate an immunedefense. It is believed that vaccination against tumors may result inprotection from tumor recurrence due to immunological memory.

Recently, interest in immunotherapies based on cancer vaccines hasprompted attempts to develop such vaccines as an additional treatmentoption for cancer patients. Several types of cancer vaccines have beenconsidered, including whole cell, defined tumor antigens, peptides andDNA vaccines. Except for whole cell vaccines, all other vaccines requirea thorough understanding of the tumor antigen(s) involved. The non-wholecell vaccines, such as peptide or whole protein vaccines orantigen-specific DNA vaccines require a substantial knowledge of theexpression of those tumor antigens, and their immunogenicity in cancerpatients. Acquiring such data involves substantial investment indefining the tumor antigens. Besides, most of these types of vaccinesinvolve usage of one tumor antigen (e.g., telomerase antigen, prostaticacid phosphatase). The anti-tumor immunity is complex, and multipleantigens and multiple epitopes are likely to be involved for efficaciousclinical outcome. In almost all cancers except the ones induced byviruses, the tumor antigens are mostly self-proteins which have beentolerized during the development of the immune system, and hence it isdifficult to induce an immune response against them. In the absence of aclear cut understanding of all tumor antigens involved in breakingself-tolerance and in the induction of clinically relevant immunityagainst cancer tissue, whole cell vaccines become good candidates forpresenting a plethora of tumor antigens to the immune system, therebyhedging against tolerogenic epitopes.

Several cancer vaccines have been tested in clinical trials, eitheralone or in conjunction with various adjuvants. Unfortunately, theclinical efficacy has thus far not been impressive. Nevertheless, withrespect to defined vs non-defined (whole cell) vaccines, the whole cellvaccines appear to be superior.

Tumor antigen vaccines generally show poor antigenicity due to immunetolerance. Most require interventional therapies in order to provide anadequate “danger” signal to the immune system in order to activate arobust, clinically meaningful antitumor immunity.

The adaptive immune response to tumors alone is poor, mostly because thetarget antigens are self-proteins, except in cases of tumor induced byviruses. Since the immune system has evolved to recognizemicrobial/pathogenic organisms, it is possible that when self-antigensare presented by antigen presenting cells (APCs), there is normally no“danger” signal, and the APCs therefore provide tolerizing signals toavoid autoimmunity. In the presence of microbial antigens, such as Emmprotein, which when expressed in tumor cells, processed and presented bythe APCs, is perceived by the adaptive immune system as a “danger”signal. Mature APCs present antigens to T cells, which generateactivated MEW class I restricted CD8+ and class II restricted CD4+ cellsand results in a clinically effective anti-tumor immunity.

Several similar types of cancer vaccines have been tested inpre-clinical and clinical studies, but thus far only one cancer vaccine,Provenge® (Sipuleucel T, Dendreon Corporation), has been approved by theFDA and only for a single indication; i.e., for use in asymptomatic orminimally symptomatic castration resistant prostate cancer patients.

Provenge® is manufactured by “feeding” patient's APCs in vitro withprostatic acid phosphatase fused to GM-CSF (adjuvant) to inducematuration of APCs, which then stimulate the immune system afterinfusion into the patient. Provenge® is antigen specific and cannot beemployed universally against other types of cancers. The survivalbenefit with Provenge® averages 4.1 months (Kantoff P W et al., N Engl JMed 2010; 363:411-22).

Autologous whole cell vaccines have been reported, including GVAXvaccine for colorectal cancer. Isolated autologous tumor cells weremixed with Bacillus Calmette-Guerin (BCG) as the adjuvant andadministered to cancer patients. While some limited clinical activitywas observed, it did not reach statistical significance. There was nodifference in time to relapse or overall survival (OS). The ulcerationinduced at the vaccine site by BCG was a substantial issue.Additionally, the FDA requirement for a sterility test could not befulfilled due to the nature of the tissue leading to prematuretermination of a Phase III trial.

Some success has been reported with an autologous whole cell vaccineagainst canine lymphoma (U.S. Pat. No. 7,795,020). Cancer cells from asolid lymphoma tumor were isolated, transformed in vitro with a vectorexpressing Emm55 protein on the lymphoma cell surface, and administeredto the subject from whom the lymphoma cells were isolated.

While whole cell vaccines have been demonstrated to have clinicalactivity, manufacturing this type of vaccine requires surgical removalof patient's palpable lymph node, ex vivo processing of cells,transformation of cells with Emm protein, irradiation of tumor cells,and the quality control (QC) of the vaccine for individual patients.With solid tumors, it may be difficult to obtain sufficient cells forprocessing, and processing can require from one to several weeks.

Autologous melanoma and renal cell carcinoma (RCC) in combination withBCG have been tested in patients (Baars A et al., Ann Oncol (2000)11:965-970; de Gurijil et al., Cancer Immunol Immunother (2008)57:1569-1577). A slight improvement in 5 year OS of 33% vs 35% formelanoma and 77% vs 68% for RCC over the historic control, respectively,were observed. Unfortunately, due to ulceration at the vaccination site,BCG was disqualified. Additional trials were conducted with a BCGreplacement, hypo-methylated bacterial CpG DNA, in an attempt to proveequivalency with BCG in RCC patients. With a 20% clinical response rate,the authors concluded that equivalency was demonstrated with CpG.

Regardless of some positive results, the potential side effects of CpGcaused concern. The safety profile of CpG was investigated in rodents,nonhuman primates and humans. Safety issues included the possibilitythat CpG might increase host susceptibility to autoimmune disease orpredispose to toxic shock. The immune stimulation elicited by CpG motifscan reduce the apoptotic death of stimulated lymphocytes, inducepolyclonal B-cell activation and increase the production ofautoantibodies and proinflammatory cytokines, all of which are known toincrease the risk of autoimmune disease, especially in organ-specificautoimmune diseases. The organ-specific autoimmune diseases aretypically promoted by the type of Th1 response preferentially elicitedby CpG. For example, in an IL-12-dependent model of experimentalallergic encephalomyelitis (that mimics multiple sclerosis), animalstreated with CpG and then challenged with autoantigen developedautoreactive Th1 effector cells that caused disease, whereas miceinjected with autoantigen alone remained disease free.

A more classic vaccination approach was based on stimulation of theimmune system by administering an immunogenic foreign protein. In amolecular mimicry model, CpG was co-administered with Chlamydia-derivedantigen. Unfortunately, this promoted the induction of autoimmunemyocarditis (Bachmaier K et al., Science, 1999; 283:1335-1339). CpG alsoincreased the susceptibility of mice to interventions that can inducearthritis. These results indicated that CpG adjuvant promotes thedevelopment of deleterious autoimmune reactions under certaincircumstances. This concern was heightened when a clinical trial usingCpG as an adjuvant for the hepatitis vaccine was halted after onesubject developed Wegener's granulomatosis, an autoimmune diseasecharacterized by inflammation of the vasculature. In addition to therisk of autoimmunity, several studies noted an elevation in thefrequency and/or severity of local adverse events (injection sitereactions such as pain, swelling, induration, pruritus and erythema) andsystemic symptoms (including flu-like symptoms) by CpG-adjuvantedvaccines.

Another approach to cancer vaccines has been to mix cell lines of aselected type of cancer derived from different individuals of the samecancer. An allogeneic pancreatic cancer cell line expressing theadjuvant GM-CSF was used in a phase I clinical trial followingpancreaticoduodenoectomy. The administered cells proved to be non-toxicbut delayed type hypersensitivity (DTH) reaction in the recipients wasobserved (Jaffe, E M et al., J. Clin. Oncol. 2001, 19, 145-156).

In other studies using allogeneic cells, a prostate cancer trialemployed a mixture of 3 different allogeneic prostate cancer cell lineswith BCG. Time to progression improved from 28 to 58 weeks. Withprostate GVAX allogeneic vaccine (a mixture of LNCaP and PC-3 cell linesexpressing GM-CSF), two clinical trials were conducted—VITAL-1 andVITAL-2. In VITAL 1 trial in hormone resistant prostate cancer patientsthat compared GVAX against docetaxel, no difference between the groupswas found. The VITAL 2 trial that compared GVAX+docetaxel againstdocetaxel had to be terminated due to safety concerns. The adjuvantGM-CSF, an FDA approved drug capable of stimulating white blood cellgrowth, has been widely used in cancer vaccine trials with varyingresults.

DNA vaccines have been the subject of a few limited studies. Plasmid DNAvaccines are circular DNA encoding one or more tumor associated antigens(TAAs) and immune-stimulating or co-stimulating molecules, which areadministered intramuscularly, intranodally or intratumorally. The localtissue specific cells and the APCs at the injection site then expressthe antigens to stimulate the immune system. It is believed that thecross-presentation of antigens by the APCs will be the most importantfactor in the induction of robust anti-tumor response with DNA vaccines.While safety and efficacy of naked plasmid vaccines have been tested inonly a small number of clinical settings, at least the intramuscular,intranodal or intratumoral vaccinations have been shown to be safe andcapable of eliciting immune responses to some extent in a few patients.

Naked plasmid DNA vaccines used in clinical settings include constructscoding for TAAs. For example, in cohorts of: 1) B-cell lymphomapatients—TAA: idiotypic determinants; 2) melanoma patients TAAs: gp100,MART-1-derived peptides and tyrosinase or tyrosinase-derived peptides;3) colorectal carcinoma patients—TAA: carcinoembryonic antigen and CEA;4) HPV-16+ cervical intraepithelial neoplasia (CIN) patients—TAA: HPV-16E6; and 5) individuals affected by prostate carcinoma—TAA: prostatespecific antigen (PSA) have been tested or are being studied. While theresults of these trials are mostly not yet available, they are gearedtowards specific cancers, and cannot be used in multiple tumors becausethey depend on TAAs.

SUMMARY OF THE INVENTION

Vaccines provide protection by stimulating immunogenic responses in thebody typically generated by administering a substance consisting of animmunogenic material associated with the cause of the disease. Cancersdiffer from externally invasive bacterial, viral and fungal diseases inthat they arise in vivo from natural cells. In theory, vaccinesconsisting of tumor antigens might be useful if such antigens could beidentified for all cancer types; however, identification of theseantigens is limited.

The natural immunogenicity of a unique M type protein encoded by aStreptococcus gene was used to engineer whole tumor cells to act as animmunogenic primer for tumor antigens in vivo. The therapeutic DNAvectors of the present invention contain a Streptococcus emm gene. Forexample, emm gene from Streptococcus pyogenes serotype 55 encodes theEmm protein, an M type polypeptide that is highly immunogenic in caninesand humans and likely to be in other species. One can construct anexpression vector with an emm gene from any one of several StreptococcusM-types by operatively linking the appropriate emm gene insert with apromoter in a mammalian expression vector. The use of this type of DNAvector is exemplified with the plasmid vector pAc/emm which cantransform mammalian cells to express the immunogenic Emm polypeptide.

Using an emm DNA vector as an example, customized Emm “vaccines”responsive to a specific cancer or to several types of cancers,depending on the source and type of cancer have been formulated asdescribed herein. When the original cancer source is a solid tumor,cells are isolated from the tumor and subjected to enzymatic digestionprior to culturing and transforming the tumor cells. The transformedcells are then administered to the patient from whom the cancer cellswere isolated, thereby acting as an autologous vaccine.

Therapeutic treatment of solid tumors is exemplified with the emm DNAfamily of vaccines described here. The expression of Emm or other M typeproteins in tumor cells enhances immunogenicity. Innate and adaptiveanti-tumor immunity is activated in vivo when tumor cells transfectedwith an appropriate emm DNA vector are administered to a mammal havingthe same tumor type. The result is a clinically meaningful responseagainst tumor cells, including tumor regression or prevention ofrecurrence.

The Emm proteins, the immunogen described herein, are generally known asM type proteins. The M protein is a fibrillar coiled-coil dimer thatextends from the bacterial cell wall, and is considered an archetypalGram-positive surface protein. The M protein is a key virulence factorand an immunogen and therefore has been a major target for Group AStreptococcus (GAS) vaccine development. Many M proteins containvariable A and B repeats while all contain conserved C repeats. It isalso likely that the epitopes that reside within variable, hypervariableand conserved regions of M protein have been preserved across multiple Mproteins. Therefore, even if all the M proteins do not have the sameamino acid sequence, many of them could be used as demonstrated withEmm.

Most of the M protein sequence consists of heptad repeat motifs in whichthe first and fourth amino acids are typically hydrophobic, and are corestabilizing residues within the coiled coil (McNamara et al., Science,2008, 319). Heterogeneity in the amino acid sequence of the N terminalpart of M protein, resulting in antigenic diversity (>200), forms thebasis of GAS Emm-typing. The size of the predicted mature form of Mprotein was highly variable and the M protein sequence is heterogeneous,ranging from 229 to 509 residues. Importantly, M protein length washighly correlated with Emm pattern. For instance, M proteins of patternA-C were the longest (average 443 residues), followed by pattern D(average 360 residues), while those of pattern E were the shortest(average 316 residues 320).

An important aspect of the invention is direct vaccination with atherapeutic DNA vector. The emm DNA vector expressing an immunogenicpolypeptide is injected into a solid cancer. The DNA vector enters thetumor cells in vivo and expresses a highly immunogenic polypeptide. Thepolypeptide stimulates an immune response in vivo to that cancer, actingas an internally generated immuno-stimulant. A DNA plasmid vector suchas pAc/emm is injected into the solid tumor. The vector transforms thecancer cells in the tumor which then express the encoded Emmpolypeptide. The transformed cancer cells thus become in vivo/in situvaccines against the tumor cells. Use of therapeutic DNA vectors avoidshaving to isolate, culture and transform tumor cells in vitro becausethe vectors can be introduced directly into a solid tumor mass.

Autologous vaccines can also be prepared from the described Emmtherapeutic vectors. Cancer cells are isolated from a patient, culturedand transformed in vitro with the vector. The autologous transformedcells are administered to the cancer patient, typically by injection. Animmune response to the cancer in the patient is generated.

There are several variations of cell-based M-type polypeptide vaccineswhich can significantly improve an immune response to cancer cells. Thevaccines can be utilized to augment or enhance conventional anti-cancertreatments either as a primary or an adjuvant treatment. Given thatthere are >200 different M type proteins, variants of an Emm or M typevaccines can be produced which will generate an immune response to tumorcells.

Individualized “custom” autologous tumor vaccines can be prepared byisolating and culturing solid, liquid or metastatic tumor cells from onepatient. The cells are transfected in vitro with an emm plasmid whichexpresses Emm protein on the cell surface and in the cytosol. A vaccinecomposition prepared from the transformed cells is administered to thepatient. The immune system responds to the cancer cells as foreign.

More universal versions of Emm and other M-type vaccines can be preparedby transforming cell cultures of cancer cells isolated from differentindividuals having the same type of cancer. Allogeneic tumor specificcell lines can be transfected in vitro with emm vector to provide atumor specific vaccine for administration. The cell lines can be derivedfrom many individuals (allogeneic cell lines) to prepare vaccines bymixing 2 or more cell lines of that cancer. The mixed cancer cell linesare transfected in vitro to express Emm or other related M-typepolypeptides, formulated into a vaccine and administered to patients.The allogeneic vaccines can be readily prepared for any solid, liquid ormetastatic cancers by having cell lines from these cancer tissues, andmixing 2 or more cell lines of a specific cancer for transformation andadministration of the vaccines into cancer patients having that specifictype of cancer.

For treatment of cancer patients, the emm therapeutic vector can bedirectly administered into the tumor; alternatively, any stage or gradeof cancer can be treated with allogeneic whole cell vaccines, so long asimmune system has not been compromised, preferably in conjunction withchemotherapies that are known to induce immunogenic cell death of cancercells. In patients with lower grade and early stages of cancer, vaccinescan be used as mono therapy.

With the direct DNA vaccine, injections can be administered directlyinto tumor lesions using lipid reagents, needless injectors,multi-needle administration patches, in vivo electroporation, J-tip,into palpable tissue, or visceral tumor lesions with the guidance ofcomputed tomography (CT) or ultrasound. In using the directadministration of the DNA vector, there is no need to harvest tumorcells from the patient. Processing the cells, transformation andirradiation steps are not required so that expense is reduced andefficiency increased compared with the in vitro process of preparing thecancer vaccine ex vivo.

There are several advantages to the use of allogeneic vaccine cells. Theuse of antigenically/cytogenetically well-defined cell lines providesaccess to a sustained and virtually limitless source of TAAs. Cell linescan be highly standardized and are suitable for large-scale productionof allogeneic vaccines. Single batches of allogeneic vaccines for allvaccines, independent of HLA haplotype, eliminates variability in thequality and composition of the vaccines and facilitates reliablecomparative analysis of clinical outcome, and also eliminates the needfor continuous production of tailor-made individual vaccines. Overall,batch production of allogeneic vaccines simplifies the logistics,reduces the labor intensity of vaccine production, simplifies qualityassurance processes, delivery process, and increases cost-effectiveness.

Vaccines produced from transformed allogeneic cell lines will have broadapplication both prophylactically and therapeutically. For a universalEmm vaccine, cell lines can be developed from solid tumors, liquidtumors, metastatic or circulating cancer cells and can be derived frommany different individuals. A cocktail of the different transformedcancer cell lines can be formulated into a vaccine for that particularcancer indication. Such a heterologous Emm vaccine can be prepared fromthe same cancers or different types of cancers from several subjects.

The described customized and individualized vaccines provide a tool forpersonalized medicine because an Emm vaccine can be developed for aspecific cancer in a subject. As a tumor evolves into antigenicallydistinct cell types due to a progressive accumulation of mutations,tumor cells can evade existing antitumor immunity that had beenpreviously induced by initial vaccine treatment. In such situations,additional vaccines targeting those tumor cells can be readily produced.

Checkpoint inhibitors are antibodies or siRNAs that can be developed andused to revert immune exhausted T cells into activated T cells withinthe tumor bed that would be clinically beneficial. Agonistic antibodiesor immuno-stimulants are reagents that can augment anti-tumor immunity.The disclosed Emm cancer vaccines can also be used in conjunction withmonoclonal antibodies to checkpoint inhibitory molecules such as CTLA-4,PD-1, PD-L1, PD-L2, LAG3, TIM3, TIGIT, antibodies to costimulatorymolecules such as CD40, OX40, antibodies capable of regulating T regssuch as anti-GITR and pan anti-BCL-2, or cytokines such as IL-2, TNF-α,IFN-γ, IFN-β, and TLR agonists.

A unique transmembrane (TM) sequence is located within the anchor regionof the Emm55 protein. While this TM sequence is present in many other Mproteins, it is not present in other proteins or in other species. Thisunique TM can anchor the Emm55 protein on mammalian cell surfaces (FIGS.4 and 5). Therefore, the TM sequence described here can be used forsurface expression of proteins in general, and specifically, when highlevels of secreted proteins prove toxic or when stable cell surfaceexpression is considered necessary for stimulation/activation of cells.For example, by limiting expression only to the cell surface using TMsequences, membrane bound GM-CSF and TNF-α have demonstrated anti-tumoractivity without cytokine mediated toxicities. In general, TM regionsare membrane-spanning domains that consist of a continuous stretch of20-30 nonpolar residues with a predominance of aliphatic side chains atthe center and aromatic residues at both ends. The TM forms the basicstructure for anchoring cell surface proteins in the cell wall or plasmamembrane. TM regions are also responsible for egress from the cytoplasmto the surface. Surface proteins represent more than 50% of allcurrently available drug targets. About 25-30% of all proteins arelocated on membranes. In general, TM domains can be classified intoß-sheet barrels or as α-helical forms, both in prokaryotes andeukaryotes. In α-helices, the main chain amides are all locallycomplemented so that the surface contacting the nonpolar membraneinterior is exclusively formed by the nonpolar side chains, suggestingthe usefulness of an α-helix as a membrane-crossing element. While theproteins of the cytoplasmic membrane consist mostly of TM α-helices, theß-barrel TM proteins are found in outer membranes of gram-negativebacteria, the cell wall of gram-positive bacteria, and outer membranesof mitochondria and chloroplasts.

Proteins are targeted to the endoplasmic reticulum (ER) membrane throughan N-terminal TM region. With the presence of a peptidase cleavage sitedownstream of the TM, the protein has enhanced exit capabilities fromthe ER, and with the further addition of a second TM region, the proteincan efficiently anchor and accumulate in the plasma membrane. Surfaceproteins can be grouped as Polytopic, with multiple membrane spanningdomains; e.g., G proteins and Bitopic, with a single membrane spanningregion; e.g., receptor tyrosine kinases, immunoglobulin superfamilyreceptors, integrins, plexins, syndecans, neuropilins, and cadherin).

The entire anchor region of Emm55 protein, which includes the TM domain,can be described based on the deduced structure of M6, the M proteinfrom another serotype of Group A Strep, i.e., a Gram positive bacterialcell wall protein similar to Emm55. The N-terminal portion of theprotein is exposed outside the wall as coiled coil α-helical dimer rodscontaining sequence repeat blocks. The anchor region which “anchors” theprotein to the cell wall contains Pro/Gly followed by a 19 AAhydrophobic region. A highly conserved heptad peptide motif, LPXTG, islocated within this region (FIG. 18). The α-helical TM domain is locatedwithin this anchor region as shown in the Emm55 primary sequence (FIG.16, SEQ ID NO: 5).

The prokaryotic TM domain of Emm55 is expected to be useful particularlyin the design of fusion products. Examples would include the ability to:

Create synthetic fusion product(s) from non-trans-membrane proteins.

Create fusion products from biologically active compounds, proteins, ordrugs in order to disrupt functional intracellular or othertransmembrane activities.

Create fusion products from biologically active compounds, proteins, ordrugs to enhance functional intracellular and extracellular activities.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic showing production of three different types of Emmvaccines.

FIG. 2 illustrates the process for preparation of an Emm autologouswhole cell vaccine.

FIG. 3 shows the process for preparation of an Emm plasmid DNA vaccine.

FIG. 4 shows the process for preparation of an Emm allogeneic whole cellvaccine.

FIG. 5 compares IgG antibody levels before treatment, after 4 injectionsand after 8 injections.

FIG. 6 shows the primary mammary tumor burden in canine patient. Overalltumor burden was stable until the 5^(th) vaccination with only 11%change in tumor mass, after which, at the time of 8^(th) injection,tumor size had increased by 33%, thus becoming a progressive disease.

FIG. 7 shows a quality of life (QOL) assessment by the pet owner. Thenumber on the x-axis represent 3^(rd), 5^(th) and 8^(th) vaccinationtime points at which QOL assessments were provided.

FIG. 8 shows that CD45+ hematopoietic cells were present along withtumor cells. More than 33% of the cells were CD45+ cells, suggesting arobust infiltration of immune cells into the tumor.

FIG. 9 shows the presence of T and B cells in the primary canine mammarytumor.

FIG. 10 shows that the antibody titer to tumor cells increasedvaccination. In all three dilutions of the plasma, the antibody levelsare significantly higher than pre vaccination plasma.

FIG. 11 shows regression of tumor lesions during the post vaccinationregimen. Vaccine doses were administered on days 1, 19, 55, 79, 110,145, 187, and 255. Responses were studied until the 289^(th) day.Lesions were measured prior to injections. All index lesions weremeasured, and the percent of reduction of tumor size was calculated forinjected and noninjected lesions before treatment (black bars) and 2weeks after the eighth vaccination (gray bars). The percent reductionsobserved during the post vaccination regimen are indicated above thegray bars. Reductions ranged from 19% to 55% of the original tumor size.

FIG. 12 shows augmentation of antimelanoma antibodies during thevaccination regimen. Vaccine doses were administered on days 1, 19, 55,79, 110, 145, 187, and 255. Responses were studied until the 289^(th)day. Antibody levels were determined using ELISA. The lysate proteinfrom a noninjected melanoma specimen was used as the antigen source at10 μg/mL. The ELISA was developed using goat anti-equine IgG antibodiesconjugated to AP enzyme and PNPP substrate. The IgG antibody level inthe plasma, at a 1:160 dilution in the ELISA assay, was 2-fold increasedand sustained over the course of the therapy. The error bars show theSEM of triplicate values. AP, Alkaline phosphatase: ELISA, enzyme-linkedimmunosorbent assay: IgG, immunoglobulin G: PNPP, p-nitrophenylphosphate.

FIG. 13 shows a nucleotide sequence alignment between the subjectapplication (Query, SEQ ID NO: 1) and the emm55 sequence (Sbjct, SEQ IDNO: 3). The differences have been indicated by the boxes. Transversionmutations=7. Transitional mutations=9. Insertional mutations in Query1=4. Deletional mutations in Query 1=1.

FIG. 14 shows an amino acid sequence alignment between the subjectapplication (UserSeq1, SEQ ID NO: 2) and the original emm55 sequence(UserSeq2, SEQ ID NO: 4). 97.1% identity in 552 residues overlap; Score:2591.0; Gap frequency: 0.4%.

FIG. 15 shows the priming of antibody response by allogeneic whole cellvaccine.

FIG. 16 is an Emm55 protein primary sequence diagram showing the TMregions of the surface expressed protein.

FIG. 17 is a Western blot showing expression of Emm55 in a kidney cellmembrane.

FIG. 18 shows the structure of the M6 protein.

FIG. 19 shows flow cytometric analyses of surface expression of Emm55 on˜20% of transfected cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides Emm therapeutic DNA vaccines and methodsfor use in stimulating anticancer immunity in cancer patients.Exemplified are Emm DNA therapeutic vector injections into solid tumorsin vivo and several variations of whole cell Emm vaccines, includingautologous and allogeneic vaccines.

The described methods relate to the generation of whole cell tumorvaccines, prepared either in vitro (Emm expressing autologous orallogeneic cells) or generated in vivo/in situ (emm plasmid DNAadministered intratumorally). The whole cell vaccine has the advantageof activating anti-tumor CD8+ T cells via direct and indirect(cross-priming) pathways. Whole tumor cell vaccines are consideredsuperior to other types of vaccines because they present a plethora oftumor antigens (known and unknown) to the immune system. An overview ofmetastatic colon cancer immunotherapy trials has demonstrated a higherclinical benefit rate with whole cell vaccine (46%) compared todendritic cell based (17%), peptide (13%) or idiotype antibody based(3%) vaccines. In a meta-analysis of tumor vaccines encompassingmultiple cancers, Neller et al. (Seminars in Immunology, 2008, 20:286-295) concluded that whole cell tumor vaccines provide objectiveclinical responses in 8.1% of patients compared to that of 3.6% withdefined antigens.

In general aspects, the invention relates to methods designed to promotean immunogenic response to whole tumor cells using a therapeutic emm DNAvaccine. The vaccine can be used as autologous whole cell vaccines,direct plasmid vaccines given intratumorally, and as allogeneic wholecell vaccines.

It is also likely that the epitopes that reside within variable,hypervariable and conserved regions of M protein have been preservedacross multiple M proteins. At least 2 known epitopes described in theliterature are found in both the Boyle emm55 and other emm sequences.For example, two sequences of an EMM protein described as immunogenicepitopes in the mouse are conserved in both sequences (highlighted as“mouse epitope”). A potential linear B cell epitope described in humansuses M1 (EMM1) peptides which react with sera. When aligned, the M1sequence has only 5% coverage of emm sequence suggesting substantialdifferences between both genes. However, there is 55% identity at theepitope region (highlighted as “potential human epitope”) in both Emm55and Emm sequences. Therefore, although all the M proteins do not havethe same amino acid sequence, many of them will have preservedimmunogenic epitopes.

The natural immunogenicity of Emm proteins is important in theformulation of the described Emm vaccines. The expression of the proteinin tumor cells greatly increases tumor cell immunogenicity, which inturn promotes activation of both innate and adaptive antitumor immunity.A clinically meaningful response against tumor cells, such as tumorregression/prevention of recurrence results from in vivo activation ofthe immune system.

Due to the nature of the coevolution of microbes and the immune system,with constant selection pressure applied bilaterally, microbial productshave special impact on the innate and hence adaptive immune system viapathogen recognition receptor (PRRs) and toll like receptors (TLRs). Bythese mechanisms, the microbial products can deliver the required“danger” signal, and robustly activate the immune system. Therefore, theEmm based cancer vaccines are more capable of inducing a clinicallymeaningful antitumor immunity.

S. pyogenes bacteria are recognized as foreign by APCs, includingdendritic cells (DCs). The innate immune system is activated through acoordinated interplay between MyD88-independent events involvingphagocytosis and antigen processing as well as MyD88-dependent signalingpathways involved in the induction of DC maturation and cytokineproduction. DCs deficient in signaling through each of the TLRs reportedas potential receptors for gram positive cell components, such asToll-like receptor (TLR)1, TLR2, TLR4, TLR9, and TLR2/6, are notimpaired in the secretion of pro inflammatory cytokines and theupregulation of costimulatory molecules after S. pyogenes stimulation.This implies a multi model recognition in which a combination of severaldifferent TLR-mediated signals are critical. Emm protein may alsoinitiate a vigorous immune response by activating innate immunity via aTLR system when tumor cells are modified to express Emm in cancerpatients in situ by direct emm vector administration intratumorally, oradministered as whole cell cancer vaccines, either as autologous orallogeneic.

The ability of Emm protein to activate innate immunity and adaptiveanti-tumor immunity makes this protein an ideal base for producingautologous (patient's own) tumor cells modified to express Emm.Manufactured emm gene-containing plasmid DNA can be administereddirectly into a tumor (in situ). Emm-expressing disease-matched, pooledcancer cells can be prepared from defined allogeneic cancer cells. Thesetypes of Emm vaccines will aid in the management of multiple cancers andcan be used in conjunction with conventional standards of care, both inhumans and in veterinary patients.

A highly important factor that will determine outcome for any cancervaccine in the clinical setting lies in an ability to clear the tumorbed of the immunosuppressive environment created by tumor/secretedfactors. Tumor microenvironment in multiple cancers is occupied by cellsthat are known to attenuate antitumor responses, such as T regulatory (Tregs) cells, tumor associated macrophages and myeloid derived suppressorcells.

Such situations often result in the appearance of vaccine-inducedimmunity in the periphery, but not in the tumor bed, and hence areclinically irrelevant. Because Emm protein is a microbial product, itimproves immunity by inducing the ingress of vaccine-activated APCs intothe tumor draining lymph nodes and alerts the immune cells residing inthat lymph node with a “danger” signal. All three described Emm vaccinemethods are effective in this process. The various Emm treatment methodsdescribed will aid in the management of multiple cancers along withtheir standard of care, both in humans and in veterinary patients. Themethods for producing and using the Emm vaccines are applicable fortreatment of all solid and liquid tumors that can be surgically removed(autologous and allogeneic) or that can be accessed, either by palpationor by using equipment such as ultrasound and CT (direct DNA).

The disclosed Emm cancer vaccines can also be used in conjunction withmonoclonal antibodies to checkpoint inhibitory molecules such as CTLA-4,PD-1, PD-L1, PD-L2, LAG3, TIM3, TIGIT; antibodies to costimulatorymolecules such as CD40, OX40; antibodies capable of regulating T regssuch as anti-GITR and pan anti-BCL-2; or cytokines such as IL-2, TNF-α,IFN-γ, IFN-β, and TLR agonists. The emm vector can also be usedcomprising additional nucleic acid that expresses immunologic moleculessuch as cytokines IL-2, IL-12, IL-18 and MHC genes. These will augmentanti-tumor immunity.

Materials and Methods

The original emm55 gene (SEQ ID NO: 3), expresses a protein derived fromthe M serotype 55 group A streptococci isolate A928, termed “Emm55 orM55”, which has an amino acid sequence typical of M-like proteins. TheN-terminal variability of M proteins generates more than 200 distinctgenotypes as determined by molecular typing. Due to theirimmunogenicity, M proteins have also been considered as vaccine targetsagainst GAS infections.

The S. pyogenes emm gene used in the examples illustrated here codes foran expressed Emm protein and includes the signal and anchoring sequences(SEQ ID NO: 1). The original emm55 gene sequence (SEQ ID NO: 3) waspublished by Boyle, et al. in 1995 (Molecular Immunology. 32: 9,669-478).

FIG. 13 and FIG. 14 show a comparison between the Boyle's publishedemm55 gene and the emm gene used in the examples described herein.

The emm nucleotide sequence used in the DNA vector examples exhibitshigh identity to Boyle's emm55 sequence and high identity in theexpressed protein sequence. Despite high identity, the protein expressedfrom the disclosed DNA vector has an additional 15 amino acids of theplasmid frame work inserted into Emm protein due to a missense mutationof stop codon TAA to GAA in emm gene.

EXAMPLES

The following examples are provided as illustrations of the inventionand are in no way to be considered limiting.

Example 1. Emm Transfected Lymphoma Cells as Vaccines

This example illustrates use of an Emm autologous whole tumor cellvaccine. The vaccine was prepared from tumor cells isolated from acanine lymphoma patient. The cells were then modified/transformed invitro with a pAc/emm plasmid DNA before formulation into a vaccinecomposition and used for treatment.

A 6 year old intact male German Shepherd, was diagnosed with caninelymphoma in late March 2012. He first presented with generalizedlymphadenopathy; and the lymphoma diagnosis was confirmed via fineneedle aspirates of the prescapular lymph nodes. Due to an intoleranceof prednisolone and other traditional medications, the owner electedautologous vaccine therapy. In late April 2012, a prescapular lymph nodewas surgically removed and submitted for histopathology and autologousvaccine preparation. The histopathology results indicated high gradelymphosarcoma with a guarded prognosis for survival. From the remaininglymph node tissue, 2.9×10⁹ cells with 100% viability were collected.Forty eight hours post transfection, 86×10⁶ viable transfected cellswere collected and irradiated. Eight vaccine doses were prepared foradministration.

In late April 2012, the Shepherd received the first four vaccinesintradermally once weekly for 4 weeks. After administration of each ofthe first four vaccines, the owner reported an increased energy levelthat lasted 5-6 days. At the request of both owner and clinician, theShepherd received the last four vaccines intradermally every 2 weeks. Atthe time of the 7^(th) vaccine, owner reported that his appetite andenergy level had improved. The Shepherd received the eighth vaccineearly July 2012. Two weeks later, the clinician reported that hisappetite was “waxing and waning” and his energy level was moderate.

The Shepherd died Oct. 5, 2012. The clinician reported that thetraditional prognosis for this dog was only 4-6 weeks with no therapy.Thus, the overall survival was prolonged by 6 months. Based on theclinician's assessment, an improved QOL was achieved during theremaining 6 months of his life. The improved survival and QOL wereassociated with the induction anti-tumor antibody responses (FIG. 5).

Evidence of induction of an anti-tumor immune response was determinedusing autologous whole cell lysate in an Enzyme-Linked ImmunosorbentAssay (ELISA). The purpose of the ELISA was to determine the titer ofthe anti-tumor antibodies present in a sample and if so, how much.ELISAs were performed in 96-well plates which permits high throughputresults.

The bottom of each well was coated with tumor proteins, which bind theantibodies to selected proteins. Proteins from the Shepherd's tumorswere used. Whole blood was centrifuged out to obtain the clear plasma, asource of antibodies. When the enzyme reaction was complete, the platewas placed into a plate reader and the optical density determined foreach well. The color produced was proportional to the amount of primaryantibody bound to the tumor proteins on the bottom of the wells.

The ELISA test results using the Shepherd's blood showed that theantitumor antibody response of IgG isotype was induced and continued tobe boosted by the injections of processed autologous cancer cells,indicating an active ongoing anti-tumor immunity.

Example 2. In Vivo Emm DNA Vector Delivery into a Solid Tumor

In contrast to the whole cell Emm vaccine in Example 1, an emm plasmidwas delivered intratumorally in this example. This type of therapeuticDNA vector vaccine is applicable in multiple types of cancers to inducerobust anti-tumor immunity. The following illustrates the preparationand use of a naked plasmid DNA vaccine.

Mammary adenocarcinoma cells were transformed in vivo by intratumoraldelivery of a DNA vector expressing Emm protein.

A female Golden retriever mix afflicted with mammary adenocarcinomabecame a candidate for Emm-based plasmid vaccine under the supervisionof her attending veterinarian. She was 6-10 years old (exact ageunknown) and weighed 69 lbs. Her expected longevity was 3 months. Shehad a tumor mass (32.3 mm×30.8 mm) on the left cranial thoracic glandand several lesions on the lungs as per X-ray. This patient wasterminally ill with a huge tumor burden at the primary site (mammarygland) and several metastasized lesions in the lungs. The patient didnot receive prior or concurrent therapy.

The Retriever was injected intratumorally with 300 μg of pAc/emm plasmidDNA using a needless injector in 600 μL of endotoxin/nuclease freewater. Due to the large size of the mammary tumor, vaccination wasequally divided and administered into three different sites of the sametumor. A total of eight intratumoral injections were given at two weekintervals. Tumor measurements were made prior to each injection timepoint. No attempt was made to include measurement of lung lesions as perthe owner's request due to cost.

Blood samples were obtained at several pre and post injection timepoints in order to determine antibody and T cell responses. Three weeksafter the eighth vaccination, the tumor mass was surgically removed andprocessed.

Although the overall tumor burden increased over time, this was in partdue to the presence of tumor infiltrating lymphocytes (see below) (FIG.6).

It is known with immunotherapies that tumor masses may increase in sizeand then regress. An X-ray taken two weeks after the last vaccineinjection showed increased lung lesions. However, patient QOL, asassessed by the owner, at third, fifth and eighth injection time pointsremained consistently good (FIG. 7). The resected tumor mass from theprimary site of the mammary tumor (three weeks after the eighthinjection) was digested with enzyme mix and assessed for hematopoieticcells using anti-canine CD45-FITC conjugated antibody.

In the processed tumor cells, more than 33% of the cells were CD45+(FIG. 8). Immunohistochemistry of the tumor tissue also demonstrated thepresence of both T and B cell in the tumor and stromal (FIG. 9). Bycomparison, in one published seminal study, human breast cancer patientswhose tumor contained >5% of intratumoral CD45⁺ cells exhibited improvedprognosis. While it is difficult to correlate human observations withthat of canine mammary tumors without a well-designed clinical trial,the data show that injections with Emm55-based plasmid DNA vaccinestimulated the immune system, and that immune cells had migrated to theprimary tumor.

Preliminary analysis of the antibody response to the primary mammarytumor cells demonstrated increased antibody levels (FIG. 10). TheRetriever was euthanized 6 months post initiation of vaccine therapy dueto a breathing issue (laryngeal hemiplegia) and seizures. No postmortemdiagnosis was carried out to determine the causes of seizures andlaryngeal hemiplegia at the pet owner's request. The digested lungmasses contained <2% CD45+ cells. Taken together, the Retriever'soverall survival was significantly extended, three months more than shewas initially assessed to live without treatment. Her QOL remained goodfor five months.

This case illustrates several important factors to take into account.First, one needs to consider the initial tumor burden. In the GoldenRetriever's case, the burden not only was excessive at the tumor sitebut also showed metastasized lesions. Increased survival might beexpected with earlier detection.

It is also noted that the vaccine dose used in this example wasarbitrarily chosen based on an earlier study in an equine.

Example 3. In Vivo Delivery of Plasmid Expressing Emm into MultipleLesions

This example illustrates the transformation/modification of tumor cellsin situ through intratumoral delivery into multiple melanoma lesions.

A 19-year-old castrated male Arab/Quarter horse presented with anextensive history of cutaneous melanoma that had metastasized to theprescapular lymph nodes. At eight years of age, histopathology confirmedhis presenting lesions to be melanoma. For the next four years, thepatient's disease was stable. Between twelve and sixteen years of age,the number of this patient's melanoma lesions increased and were treatedwith cimetidine from time to time. He was also given an injection of anundefined vaccine from Canada at age fourteen. Tumor lesions wereremoved from the left flank, right hip, and right neck. However, by theage of sixteen, melanoma recurred at one of the prior surgical sites,and new cutaneous lesions were observed on his tail and neck, within hismane, and at other sites including the perianal region. Some of theselesions progressed to open sores that secreted a dark fluid exudatewhich later was shown to contain malignant melanocytes.

Several years prior to treatment with the therapeutic emm DNA vector,the patient was again treated surgically by removing both cutaneouslesions and lesions in the genital region. Results from blood workperformed in June 2010 were normal except for a high level of lactatedehydrogenase (LDH), 569 U/L. As the severity of the disease progressed,the option for further surgery was declined, and other treatments weresought. By March 2011, no reduction of tumor burden had been noted, andthe prescapular lymph nodes were beginning to become enlarged. Bloodanalysis completed in May 2011 indicated a high LDH level of 606 U/L,implying that the disease was still progressing. By June 2011, thepatient developed a fever, and the prescapular lymph nodes continued toenlarge. The exudate fluid removed from the lymph nodes revealed highnumbers of melanocytes and was contaminated with bacteria. In August2011, the horse's illness progressed still further.

The melanoma continued to progress, and euthanasia was considered.Considering the failure of all previous treatments to control or haltthe progression of the melanoma, as well as the severity and late stageof the disease, it was determined in December 2011 that the horse was acandidate for an experimental treatment that involved the directinjection of a DNA cancer vaccine.

Under strict veterinary supervision and control, the horse was treatedintratumorally with a plasmid DNA vaccine. The plasmid vector pAc/emmconsisted of a mammalian expression vector backbone and the 1.6-kb emmgene insert. The rationale behind the intratumoral injection of theplasmid DNA was to modify/transform melanoma tumor cells in situ. Theexpression of this bacterial protein on the surface of tumor cellsovercomes the inherent self-tolerance to tumor antigens.

The treatment, which commenced in December 2011, involved the directinjection of plasmid DNA into cutaneous lesions. Three visible tumormasses were selected for treatment: 1 on the right side of the neck, 1on the right side of the rump, and 1 on the tail. Two masses, one in theregion of the mane and one on the tail, served as control lesions anddid not receive the DNA cancer vaccine. The former three melanomalesions were each injected with 100 μg of pAc/emm, for a total of 300 μgof plasmid DNA in a volume of 200 μL of endotoxin and nuclease-freedistilled water, using a needless injector system at each vaccinationtime point. A total of eight plasmid DNA vaccinations were administeredto each of the three lesions. The vaccine doses were administered ondays 1, 19, 55, 79, 110, 145, 187, and 255. Responses were studied untilthe 289th day. The lesions were measured prior to injections.

The Syrijet injector is a precision instrument safely used for applyingoperative and surgical anesthetic for dental procedures. This was thefirst time this device was used for intratumoral delivery of a DNAtherapeutic vaccine.

There were no inflammatory reactions at any of the injections sitesthroughout the course of the treatment. The size of the tumor lesions(both injected and noninjected) were measured before and after each ofthe DNA cancer vaccine administrations. Assessment of tumor size wascarried out following published guidelines for the evaluation of immunetherapy activity in solid tumors.

At the baseline and subsequent tumor assessments, all indexed lesionswere measured, and the sum of the products of the two largestperpendicular diameters (SPDs) was calculated. The SPDs of the lesionswere added together to provide the total tumor burden. Table 1 shows allindividual measurements of indexed lesions (injected and noninjected).As shown in Table 1, there were fluctuations in the size of individualtumors, which may be due to the ingress and egress of immune cells butresulted in an overall reduction in tumor burden by the conclusion ofthe treatment. FIG. 11 shows the pretreatment and post measurement ofindividual indexed lesions along with observed percent of reductions inthe mass above each bar.

Table 2 summarizes the total reduction in injected and noninjectedlesions at the conclusion of the treatment regimen. Based on these data,the SPD of all injected lesions was reduced by 40.3% and that ofnoninjected lesions was reduced by 47.6%, with an overall reduction intumor burden of indexed lesions by 42.3%. When all tumor measurementswere combined from both the 3 treated lesions and the 2 untreatedlesions, pre- and post-vaccinations, the reduction observed in tumorsize reached statistical significance in a paired t test (P=0.0272).

During the course of this study, it was determined that all of the tumormasses observed stabilized, regressed in size, and ceased leaking thedark melanocyte-containing exudate. The consistency of several of themelanoma lesions went from firm to soft. By the end of September 2012,the patient had gained weight, was alert, and was healthy enough to beridden.

TABLE 1 Measurement of tumor lesions during the study period InjectedLesions (mm) Noninjected Lesions (mm) Measurement Neck Rump Tail ManeTail 1 (pre-treatment) 40 160 160 31 112  2 30 144 90 ND ND 3 30 120 157ND ND 4 26 140 83 ND ND 5 30 250 140 30 87 6 30 168 75 ND ND 7 23 223105 26 45 8 19 123 112 30 50 9 (post-treatment) 20 105 90 25 50 ND, notdone

TABLE 2 Regression of injected and noninjected tumor lesions TumorLesions Pre-treatment Post-treatment % Reduction Injected 360 mm 215 mm40.3 Noninjected 143 mm  75 mm 47.6

In order to ascertain whether the reduction in tumor burden observedcorrelated with the development of an antitumor immune response,antibody levels were measured using a standard ELISA. Data shown in FIG.12 indicate that vaccination by direct injection of the DNA cancervaccine resulted in the induction of anti-melanoma IgG antibody responsewhich increased 2-fold over time and persisted until the end of thestudy.

This example demonstrates a significant clinical benefit from injectingseveral melanoma tumors with the Emm therapeutic DNA vector. Themelanoma cells in the injected mass expressed the Emm polypeptide,became immunogenic and elicited a measurable anti-melanoma immuneresponse and systemic tumor regression contributing to prolongedsurvival.

Example 4. Allogeneic Whole Cell Vaccine Treatment of Carcinoma

A male neutered 11 year old Shetland Sheepdog was presented toMorphogenesis on May 25, 2012 for whole cell vaccine therapy. He hadpreviously been diagnosed with nonpapillary transitional cell carcinoma(CTCC) on May 4, 2012. His initial treatment included Piroxicam, anon-steriodal anti-inflammatory agent. For vaccine preparation, hisbladder mass was surgically removed and submitted for autologous tumorcell processing. Due to difficulties in early expansion of autologouscarcinoma cells, with the owner and his attending clinician's consent,an allogeneic vaccine therapy was considered for administration.

A previous patient's transitional carcinoma cells were processed andused to prime anti-tumor immunity. The 1^(st) vaccine dose contained10×10⁶ allogeneic cells. After the 1^(st) vaccine, the clinicianreported that the Sheepdog had an increased appetite and energy level.The clinician also reported that the blood in the urine had resolved,which was an issue previously. The 2^(nd) through 4^(th) vaccinescontained a mixture of both allogeneic cells and autologous cells in 80%allogeneic/20% autologous ratio. The 5^(th) through 12^(th) vaccinescontained 100% autologous cells. The first 9 vaccines were administeredonce a week for 4 weeks. He restarted receiving the 10^(th) through12^(th) vaccines biweekly. All doses were given intradermally.Approximately 1 year (July 2013) after initially started the vaccineregime, the owner reported that the Sheepdog was happy and had apuppy-like playfulness. In October 2013, the clinician reported that theSheepdog became asymptomatic of his disease and his urinary bladderappeared small and less taunt. Also in October, tumor cells werecollected from a urine specimen and expanded in culture. These urinetumor cells and original autologous tumor cells were used in combinationto supply the 13^(th) through 15^(th) vaccines, which were administered,intradermally, on a monthly basis. The 16^(th) vaccine was administeredone month after the 15^(th) vaccine and the 17^(th) vaccine wasadministered two months after the 16^(th) vaccine. The Sheepdog receivedhis final vaccine, the 17^(th) dose, in April 2014. Prior to receivingthe 16^(th) vaccine in February, the clinician reported that theSheepdog had a palpable mass in the bladder that was impinging on theanus. Due to quality of life concerns, the owner elected euthanasia afew weeks later. Though he succumbed to the disease, both clinician andowner were impressed that he survived almost two years post diagnosis.

In order to evaluate the immunogenicity of allogeneic whole cellvaccine, autologous tumor specific antibodies were measured. Theinduction of anti-tumor immune response was determined using autologouswhole cell lysate in an Enzyme-Linked Immunosorbent Assay (ELISA). Thepurpose of an ELISA is to determine if a particular protein is presentin a sample and if so, how much. ELISAs are performed in 96-well plateswhich permits high throughput results. The bottom of each well is coatedwith tumor proteins to which will bind the antibodies one wants tomeasure. In this case, proteins from the Sheepdog's tumor cells wereused. Whole blood is centrifuged out to obtain the clear plasma, asource of antibodies. When the enzyme reaction is complete, the entireplate is placed into a plate reader and the optical density isdetermined for each well. The amount of color produced is proportionalto the amount of primary antibody bound to the proteins on the bottom ofthe wells.

Based on the ELISA test results using the Sheepdog's blood, theanti-tumor antibody response of IgG isotype had been induced and whichcontinued to be boosted by the injections of autologous cancer cells,suggesting that initial 4 doses of allogeneic vaccines successfullyinduced anti-tumor immunity.

Example 5. Emm Transfected Allogeneic Cell Lines as Vaccines

The induction of robust anti-tumor immune responses is known to dependon the cross-presentation of vaccine derived TAA to specific cytotoxic Tlymphocytes (CTL) in vivo. This process of cross-priming is facilitatedby the activation of APCs such as DCs. Allogeneic cells are able topresent a viable source of TAA, which would be taken up by DC and thenpresented in the context of appropriate MHC alleles to autologous CTL.

An Emm vaccine can be prepared from transfected well-defined tumorspecific allogeneic cell lines by modifying/transforming tumor celllines in vitro with pAc/emm plasmid DNA.

Example 6. Selection of Emm Transformed Allogeneic Cell Lines

Published mouse studies have clearly demonstrated that the efficacy ofvaccines depends on the cross-presentation of vaccine derived TAA tospecific CTL in vivo. The process of cross-priming is facilitated by theactivation of professional APCs, such as DC. This suggested thatallogeneic cells will also present a viable source of TAA, which wouldbe taken up by DCs and then presented in the context of appropriate MEWalleles to autologous CTL.

For direct antigen presentation by vaccine cell lines, for example, inhumans, just by matching vaccine cell lines for the HLA-A2 and/or -A3alleles, >50% of patients should be eligible for allogeneic vaccinationregimens for a given cancer. The same is true for veterinary patients(DLA-dog leukocyte antigen, FLA-feline leukocyte antigen) and a broadapplicability can be achieved by deriving vaccine cell lines acrossbreeds to cover the wider patient population.

For indirect antigen presentation (cross-presentation) of tumorantigens, which as described above is the most efficacious way to induceimmune responses, there is no need for vaccine cells to match the MHChaplotype of the patients. By mixing 2 or more well-defined cell linesderived from patients with a given cancer, both TAAs and tumor specificantigens (TSA) can be presented by the allogeneic vaccine cells.

The cell lines derived from patients with a specific cancer type need tobe thoroughly defined, transfected with, for example, pAc/emm plasmid,selected using G418 antibiotic and cryopreserved at low passage numberto create a master cell bank (MCB). The resistance to G418 is affordedby the neomycin gene located in the pAc/emm plasmid. In the samefashion, untransfected cell lines can also be stored. The working cellbank (WCB) is then created from MCB. Cell cultures for creating vaccinescan be generated from the WCB.

The cell lines for any cancer type can be defined in terms of (as partof quality control) using the following characteristics:

Karyotype stability

Disease relevant mutations, if known

Surface phenotype

Tumor antigen profile, if known

Negative for endotoxin

Negative for mycoplasma

For humans, Negative for:

EBV, HIV, HCV, HTLV, EBV, HPV, CMV

To prepare a vaccine for a particular cancer type, two or more celllines belonging to that cancer type, which have been characterized asper quality control, can be mixed with untransfected cells in a requiredproportion. For example, 10% of transfected cell lines can be mixed with90% untransfected cell lines to give a dosage of allogeneic vaccinecontaining 10% of cells expressing Emm protein. For example, a dose canbe 10-20×10⁶ cells depending on the type of cancer and preclinicalstudies. Based on preclinical studies, the number of cell lines to beused for a selected cancer type can be fixed.

Allogeneic Emm transformed cancer cells can be shipped frozen in freezemedium or equivalents. An exemplary freeze medium is:

Veterinary Plasma-Lyte 148 (for veterinary patients)

Plasma-Lyte A (for human use)

7.5% Cryoserve

10% clinical grade heat inactivated fetal bovine serum (for veterinarypatients)

10% clinical grade heat inactivated human AB serum (for human use)

Based on data obtained from Emm autologous and emm plasmid DNA vaccines,allogeneic vaccines are able to induce anti-tumor immune response,improve survival and quality of life of the patients.

Despite variations between the emm55 protein and the disclosed emm innucleotide sequence (98% identity) and protein sequence (97.1% identityexcluding 15 amino acids of the plasmid frame work), at least 3 knownepitopes described in the literature are found in the both sequences.For example, the sequences of the Emm protein described as immunogenicepitopes in the mouse are conserved in both Emm and Emm55 sequences(highlighted as “mouse epitope”). A potential linear B cell epitope wasdescribed in humans using M1 (EMM1) peptides which reacted with sera.When aligned, the M1 sequence has only 5% coverage of Emm55 sequencesuggesting substantial differences between both genes. However, there is55% identity at the epitope region (highlighted as “potential humanepitope”) in both Emm and Emm55 sequences. Therefore, variations foundbetween Emm55 and the Emm demonstrated herein, such variations do notsignificantly alter immunogenicity of the protein. Instead.

Table 3 shows that the Emm polypeptide expressed by the vector used inthe examples is different from the polypeptide expressed by the emm55gene.

TABLE 3 Amino acid position Emm SEQ ID NO: 2 Emm55 SEQ ID NO: 4 10Tyrosine (Y) aromatic Aspartic acid (D) hydrophobic negatively charged42 Serine (S) polar neutral Asparagine (N) polar neutral 67Phenylalanine (F) aromatic Valine (V) nonpolar hydrophobic aliphatic 154Lysine (K) positively charged Threonine (T) polar neutral 174 Alanine(A) nonpolar aliphatic Valine (V) nonpolar aliphatic 188 Valine (V)nonpolar aliphatic Alanine (A) nonpolar aliphatic 195 Glutamic acid (E)negatively Serine (S) polar charged neutral 196 Arginine (R) positivelycharged Alanine (A) nonpolar aliphatic 455 Alanine (A) nonpolaraliphatic Threonine (T) polar neutral 459 Glutamine (Q) polar neutralLysine (K) positively charged 490 Valine (V) nonpolar aliphatic Alanine(A) nonpolar aliphatic 494 Threonine (T) polar neutral Lysine (K)positively charged 512 Threonine (T) polar neutral — 525 Alanine (A)nonpolar aliphatic Threonine (T) polar neutral 544 Alanine (A) nonpolaraliphatic — Amino acids found only in Emm 552 Glutamic acid (E)negatively charged 553 Alanine (A) nonpolar aliphatic 554 Glutamic acid(E) negatively charged 555 Phenylalanine (F) aromatic hydrophobic 556Cytosine (C) polar neutral 557 Arginine (R) positively charged 558Tyrosine (Y) aromatic hydrophobic 559 Proline (P) polar neutral 560Serine (S) polar neutral 561 Histidine (H) positively charged 562Tryptophan (W) aromatic hydrophobic 563 Arginine (R) positively charged564 Proline (P) polar neutral 565 Arginine (R) positively charged 566Leucine (L) nonpolar aliphatic

Example 7. Primary Sequence of Emm55 Protein Domains

The primary sequence analysis carried out using TMpred online toolavailable in SIB (ExPASY) indicates, as per statistical scores, thatthere are two potential regions of helices capable of transmembraneinsertion (Table 4). One is located within N-terminal signal peptideregion (SEQ ID NO: 7) and the other one is present within C-terminal GASanchor region (SEQ ID NO: 9). As expected, N-terminal TM helix isoriented towards inside to outside (enabling egress to surface) while TMhelix within the C-terminal anchor region is oriented towards outside toinside orientation to anchor the protein on the surface (FIG. 16).Surface and cytoplasmic expression of Emm55 protein in FIG. 16 and FIG.17.

TABLE 4Possible TM helices predicted by Tmpred (Swiss Institute of Bioinformatics)From-To Score Region Orientation score  18-36 1591within N-terminal signal region Inside to outside ++ 524-543 1385within C-terminal anchor region Outside to inside ++ Transmembraneprediction score: only scores above 500 are considered significant.Orientation score: ″++″ symbol indicates a strong preference of thisorientation. Predicted C-terminal TM sequence: FFTAAALTVMATAGVAAV (SEQID NO: 9)

Example 8. In Vitro Expression of Emm55 Protein

In vitro transfections of eukaryotic cells were used to assess theability of pAc/emm55 to drive transcription and translation of emm55.Transient transfection of human embryonic kidney cell line HEK293T (ATCCCRL-3216) with pAc/emm55 using lipofection (Lipofectamine 2000,Invitrogen; Carlsbad, Calif.) resulted in the expression of Emm55protein on the cell membrane and in the cytoplasm. Western blot analysesof whole cell protein and subcellular protein fractions of HEK293T cellstransfected with pAc/emm55, incubated with RAB-AP01 (affinity purifiedrabbit anti-Emm55 peptide antibodies) then stained with anti-rabbithorseradish peroxidase, resulted in the detection of a bandcorresponding to the predicted molecular mass of recombinant Emm55. Insubcellular protein lysates, Emm55 is detected in both the cytoplasmicand membrane fractions (FIG. 17). No protein of matching molecular masswas detected in lysates of HEK293T cells transfected with pAc/empty.Flow cytometric analysis of pAc/emm55-transfected HEK293T cells stainedwith RAB-AP01 resulted in a consistent positive population of ˜20% whengated against a rabbit IgG negative control, an anti-rabbit-PE secondaryantibody only control, unstained cells or pAc/empty-transfected cellsstained with anti-Emm55 (FIG. 19).

What is claimed is:
 1. A membrane anchoring protein comprising the aminoacid sequence SEQ ID NO: 5 located within the N-terminal signal sequenceand the C-terminal anchor region of the protein.
 2. The membraneanchoring protein of claim 1 wherein the membrane anchoring protein is abacterial protein.
 3. The membrane anchoring protein of claim 1 whereinthe helical N-terminal signal peptide region of the protein orientsinside to outside to provide egress to the cell membrane surface.
 4. Themembrane anchoring protein of claim 1 wherein the helical C-terminalanchor region is oriented outside to inside orientation on the cellmembrane.
 5. The membrane anchoring protein of claim 1 wherein the aminoacid sequence within the N-terminal signal sequence has the amino acidsequence of SEQ ID NO:
 7. 6. The membrane anchoring protein of claim 1wherein the amino acid sequence within the C-terminal anchor region ofthe protein has the amino acid sequence of SEQ ID NO:
 9. 7. The membraneanchoring protein of claim 1 wherein the protein induces an immuneresponse in vivo.
 8. The membrane anchoring protein of claim 7 whereinthe protein is a streptococcal protein.
 9. The membrane of claim 1 whichis cell membrane.
 10. The cell membrane of claim 9 which is a eukaryoticmembrane.
 11. The cell membrane of claim 9 which is a prokaryoticmembrane.